Perturbation of O-GlcNAcylation, a nutrient sensing pathway, has been demonstrated in human Alzheimer's disease (AD) brains, and inhibition of O-GlcNAcase (OGA) has been shown effective in inhibiting tau phosphorylation in animal models. This proposal focusses on the protein O-GlcNAcylation and its impact on neurological function and protein homeostasis. One of the key proteins we focus on is ?-synuclein accumulation which occurs prominently in Alzheimer's disease (AD), Dementia with Lewy Bodies (DLB) and Parkinson's disease (PD), however, whether O-GlcNAcylation is involved in ?-synuclein accumulation in vivo in mammalian models has not been investigated. This is important because targeting ?-synuclein is actively pursued as a therapeutic strategy in treating neurodegenerative disease. In our preliminary studies, we have found that inhibition of OGA increased autophagosomal and ?-synuclein accumulation and attenuated autophagic flux in primary neurons, cautioning a potential detrimental effect of inhibiting OGA, even though it was effective in decreasing tau phosphorylation. In vivo, we have shown that key proteins involved in protein trafficking are dynamically O-GlcNAcylated. In the proposed studies, we will test the hypothesis that sustained increase in O-GlcNAc causes neurological dysfunction, thereby contributing to the pathogenesis of AD and DLB. We have built a strong research team with expertise in O-GlcNAc biology, cutting-edge techniques of tag-mass spectrometry and stoichiometry of O-GlcNAc modification, autophagy, and mouse models of tissue specific OGA deficiencies. This study will: 1) Determine the impact of increased O-GlcNAc levels on neurological function in vivo. 2) Determine the mechanisms through which O-GlcNAc modification impacts neurodegeneration. The successful completion of these studies will establish a role for O-GlcNAcylation in regulating neurodegeneration, and provide new mechanistic understanding of O-GlcNAc regulation of endolysosomal function, which will guide strategies for neuroprotection. Completion of the proposed study will encourage future work introducing O- GlcNAcylation site mimetic peptide as competitive inhibitors to test the potential benefits of preventing detrimental O-GlcNAcylation while preserving beneficial O-GlcNAcylation. This proposal is directed at addressing the urgent and pressing need to understand basic mechanisms of proteotoxicity in neurons and is responsive to PAR-15-357 Understanding Alzheimer's Disease in the Context of the Aging Brain (R01).